The regiment formally mustered into service on May 13, 1864, under the command of Colonel Cyrus Reasoner and departed for Harpers Ferry, West Virginia.
On July 14, the regiment conducted a 27-mile single day's march to Hagerstown, Maryland where it took charge of a wagon train full of necessary supplies for renewed operations across the theatre.
On July 21, the regiment marched to Brown's Crossing, West Virginia and established a network of pickets and patrols along the segment of the Baltimore and Ohio Railroad running between Harper's Ferry and Martinsburg.
Shipped to the Eastern Theater, they were designed to be placed in "safe" rear areas to protect railroads and supply points, thereby freeing regular troops for Lt. Gen. Ulysses S. Grant’s push on the Confederate capital of Richmond, Virginia.
Ohio Guard units met the battle-tested foe head on and helped blunt the Confederate offensive thereby saving Washington, D.C., from capture.